Bottom Line:
As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample.We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols.The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

ABSTRACTSingle molecule localization microscopy (SMLM) techniques allow for sub-diffraction imaging with spatial resolutions better than 10 nm reported. Much has been discussed relating to different variations of SMLM and all-inclusive microscopes can now be purchased, removing the need for in-house software or hardware development. However, little discussion has occurred examining the reliability and quality of the images being produced, as well as the potential for overlooked preparative artifacts. As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample. Here we systematically investigate many of these steps including different fixatives, fixative concentration, permeabilization concentration and timing, antibody concentration, and buffering. We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols. The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

f2: SMLM microtubule images of sub-optimally fixed cells reveal sub-diffraction artifacts not observable in epifluorescence images.(A–C) Epifluorescence images of COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with PFA (A) for a shorter period than optimal, (B) at a lower concentration than optimal, and (C) for a longer period than optimal These images are not readily identified as having fixation artifacts. (D–F) Corresponding SMLM images of sub-areas from A–C show clear sub-diffraction scale damage to the microtubule architecture with some areas well preserved and continuously stained in D–E (white arrows) and other long stretches of filament missing or damaged beyond antibody-epitope recognition. Scale bars A–C: 5 μm, D: 1 μm, E–F: 500 nm.

Mentions:
Interestingly, the steps within our optimized protocols and, indeed, in all trialled protocols, that were observed to produce the most sub-diffraction scale damage were those involved in the initial application of the fixative. This included both the type of fixative used and the technique with which it was introduced into the cells. Examples of the regularly observed sub-diffraction artifacts that resulted are shown in Figure 2. Contrary to widespread practice, we observed best structure preservation when no initial washing step was conducted for the PFA and GA fixations. Application of PBS even at 37°C for 60 seconds caused changes in the clustering distribution on the MC membrane and introduced short unstained/damaged tracts on the MTs like those shown in Fig 2e. Similarly, any degree of dehydration prior to fixation caused similar artifacts to arise and because of the chamber walls, our samples were particularly susceptible to this if care was not taken. For these reasons we have specified that culture medium be removed from one side of the chamber while 37°C PFA or GA fixative is added simultaneously down the opposite chamber wall. This appears to increase the amount of non-specific aggregated stain on the coverglass outside of the cells but allows very good preservation of the MT, MC and actin substructure.

f2: SMLM microtubule images of sub-optimally fixed cells reveal sub-diffraction artifacts not observable in epifluorescence images.(A–C) Epifluorescence images of COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with PFA (A) for a shorter period than optimal, (B) at a lower concentration than optimal, and (C) for a longer period than optimal These images are not readily identified as having fixation artifacts. (D–F) Corresponding SMLM images of sub-areas from A–C show clear sub-diffraction scale damage to the microtubule architecture with some areas well preserved and continuously stained in D–E (white arrows) and other long stretches of filament missing or damaged beyond antibody-epitope recognition. Scale bars A–C: 5 μm, D: 1 μm, E–F: 500 nm.

Mentions:
Interestingly, the steps within our optimized protocols and, indeed, in all trialled protocols, that were observed to produce the most sub-diffraction scale damage were those involved in the initial application of the fixative. This included both the type of fixative used and the technique with which it was introduced into the cells. Examples of the regularly observed sub-diffraction artifacts that resulted are shown in Figure 2. Contrary to widespread practice, we observed best structure preservation when no initial washing step was conducted for the PFA and GA fixations. Application of PBS even at 37°C for 60 seconds caused changes in the clustering distribution on the MC membrane and introduced short unstained/damaged tracts on the MTs like those shown in Fig 2e. Similarly, any degree of dehydration prior to fixation caused similar artifacts to arise and because of the chamber walls, our samples were particularly susceptible to this if care was not taken. For these reasons we have specified that culture medium be removed from one side of the chamber while 37°C PFA or GA fixative is added simultaneously down the opposite chamber wall. This appears to increase the amount of non-specific aggregated stain on the coverglass outside of the cells but allows very good preservation of the MT, MC and actin substructure.

Bottom Line:
As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample.We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols.The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

ABSTRACTSingle molecule localization microscopy (SMLM) techniques allow for sub-diffraction imaging with spatial resolutions better than 10 nm reported. Much has been discussed relating to different variations of SMLM and all-inclusive microscopes can now be purchased, removing the need for in-house software or hardware development. However, little discussion has occurred examining the reliability and quality of the images being produced, as well as the potential for overlooked preparative artifacts. As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample. Here we systematically investigate many of these steps including different fixatives, fixative concentration, permeabilization concentration and timing, antibody concentration, and buffering. We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols. The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.